U.S. patent number 6,008,210 [Application Number 08/687,395] was granted by the patent office on 1999-12-28 for use of aldosterone antagonists to inhibit myocardial fibrosis.
Invention is credited to Karl T. Weber.
United States Patent |
6,008,210 |
Weber |
December 28, 1999 |
Use of aldosterone antagonists to inhibit myocardial fibrosis
Abstract
This invention discloses a method of inhibiting myocardial
fibrosis by administering epoxymexrenone at a dosage which does not
substantially increase sodium excretion by the body.
Inventors: |
Weber; Karl T. (Columbia,
MO) |
Family
ID: |
26856715 |
Appl.
No.: |
08/687,395 |
Filed: |
August 2, 1996 |
PCT
Filed: |
December 01, 1994 |
PCT No.: |
PCT/US94/13291 |
371
Date: |
August 02, 1996 |
102(e)
Date: |
August 02, 1996 |
PCT
Pub. No.: |
WO95/15166 |
PCT
Pub. Date: |
June 08, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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160236 |
Dec 2, 1993 |
5529992 |
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871390 |
Apr 21, 1992 |
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Current U.S.
Class: |
514/173 |
Current CPC
Class: |
A61K
31/585 (20130101); A61K 31/00 (20130101) |
Current International
Class: |
A61K
31/58 (20060101); A61K 31/585 (20060101); A61K
31/00 (20060101); A61K 031/58 () |
Field of
Search: |
;514/173 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
K Weber et al., "Myocardial Fibrosis, Aldosterone, and
Antialdosterone Therapy: Evolving Concepts in the Management of
Congestive Heart Failure", Department of Medicine, University of
Missouri-Columbia, pp. 1-33 (1991). .
Highlights Report #1, The XIIIth Congress of the European Society
of Cardiology (Amsterdam, The Netherlands) pp. 1-4 (Aug., 1991).
.
Remington's Pharmaceutical Sciences, 15th edition 1975, pp.
867-868. .
Doering, C.W., et al., "Collagen network remodeling and diastolic
stiffness of the rat left ventricle with pressure overload
hypertrophy," Cardiovasc. Res. 22: 686-695 (1988). .
Brilla, C.G., et al., "Remodeling of the rat right and left
ventricle in experimental hypertension," Circ. Res. 67: 1355-1364
(1990). .
Weber, K.T., et al., "Structural remodeling of myocardial collagen
in systemic hypertension: functional consequences and potential
therapy," Heat Failure 6: 129-137 (1990). .
Weber, K.T., et al., "Myocardial remodeling and pathologic
hypertrophy," Hospital Practice 26 (4): 73-80 (1991). .
Andreoli, T.E., "Introduction: Modern aspects of congestive heart
failure," Hospital Practice 26(4): 7-8 (1991). .
Brilla, C.G., et al., abstract, "Myocardial fibrosis in
experimental hypertension: potential role of fibroblast corticoid
receptors," J. Hypertension 8: S8 (1990). .
Weber, K.T., and Brilla, C.G., "Pathological hypertrophy and
cardiac interstitium: fibrosis and renin-angiotensin-aldosterone
system" Circulation 83: 1849-1865 (Jun. 1991). .
Weber, K.T., and Brilla, C.G., "Myocardial fibrosis and elevations
in plasma aldosterone in arterial hypertension," Aldosterone:
Fundamental Aspects 215: 117-120 (1991). .
Brilla, C.G., and WEber, K. T., "Prevention of mycoardial fibrosis
in hypertension: role of fibroblast corticoid receptors and
spironolactone" Proceedings of the 75th Annual Meeting of the Fed.
of Amer. Societies for Exper. Biology, 1991, abstract No. A1256,
published Apr. 21, 1991. .
American Journal of Cardiology, Klug et al., "Role of Mechanical
and Hormonal Factors in Cardiac Remodeling and the Biologic Limits
of Myocardial Adaptation," Jan. 21, 1993, vol. 71, No. 3, pp.
46A-54A. .
Journal of Molecular and Cellular Cardiology, Brilla et al.,
"Anti-Aldosterone Treatment and the Prevention of Myocardial
Fibrosis in Primary and Secondary Hyperaldosteronism," May 1993,
vol. 25, No. 5, pp. 563-575. .
American Journal of Cardiology, Brilla et al., "Antifibrotic
Effects of Spironolactone in Preventing Myocardial Fibrosis in
Systemic Arterial Hypertension," Jan. 21, 1993, vol. 71, No. 3, pp.
12A-16A. .
Journal of Pharmacology and Experimental Therapeutics, "Three New
Epoxy-Spirolactone Derivatives: Characterization in vivo and in
vitro," Feb. 1987, vol. 240, No. 2, pp. 650-656. .
Bundesverbrand D. Pharm. Ind. E. V., "Rote Liste," 1987 Editio
Cantor Aulendorf/Wurtt. .
J. Pharm. Exper. Ther. de Gasparo et al, 240(2) 650-656, 1987.
.
J. Hypertension, Brilla et al, 8(13) 58, 1990..
|
Primary Examiner: Cook; Rebecca
Attorney, Agent or Firm: Shook, Hardy & Bacon
Government Interests
GOVERNMENT SUPPORT
This invention was supported in part by grant R01-31701 from the
National Institutes of Health. Accordingly, the federal government
has certain rights in this invention.
Parent Case Text
RELATED APPLICATION
For the United States, this application is a 371 of PCT/US94/13291
filed Dec. 1, 1994 and continuation-in-part of U.S. patent
application Ser. No. 08/160,236, filed Dec. 2, 1993, now U.S. Pat.
No. 5,529,992 which in turn is a continuation-in-part of U.S.
patent application Ser. No. 07/871,390, filed on Apr. 21, 1992
abandoned.
Claims
We claim:
1. A method of inhibiting aldosterone-mediated myocardial fibrosis
comprising administering to a patient in need thereof
epoxymexrenone in a quantity that is therapeutically effective in
suppressing myocardial fibrosis without substantially increasing
sodium excretion by the body.
2. A method of inhibiting aldosterone-mediated myocardial fibrosis,
comprising administering to a human person in need thereof
epoxymexrenone in a quantity that is therapeutically effective in
suppressing myocardial fibrosis without substantially increasing
sodium excretion by the body.
Description
BACKGROUND OF THE INVENTION
This invention relates to drugs such as spironolactone which block
the activity of the hormone aldosterone, and to the use of
aldosterone-blocking drugs to prevent or treat myocardial fibrosis,
a disease condition.
In a medical context, fibrosis refers the creation of fibrotic
tissue (i.e., tissue characterized by an abnormally high quantity
of fibrous material, primarily strands of collagen). In some
situations, fibrosis is useful and necessary, such as in the
healing of wounds, but in other situations, fibrosis can be
harmful, especially when it interferes with the functioning of
internal organs. As one example, liver cirrhosis is usually
characterized by high levels of fibrosis. That condition, discussed
in the above-cited parent U.S. application Ser. No. 07/871,390, is
not directly relevant to this invention.
This invention relates to the use of mineralocorticoid antagonists
(such as spironolactone) in inhibiting myocardial fibrosis.
The correlation between mineralocorticoids and fibrosis was not
recognized prior to the work of the Applicant. However, a great
deal was known about mineralocorticoids and about fibrosis, as
separate fields in medicine and physiology. Accordingly, the
following sections provide background information on each of those
topics.
MINERALOCORTICOIDS
The adrenal glands, which sit on top of the kidneys in the human
body, are divided into two portions: the adrenal medulla (which
secretes epinephrine and norepinephrine), and the adrenal cortex.
The adrenal cortex secretes a number of hormones known as
corticoids, which are divided into two categories. Glucocorticoids
(primarily hydrocortisone, also known as cortisol) exert their
primary effects on the metabolism of glucose and other
carbohydrates; they can also secondarily retard wound healing, by
interfering with inflammatory cell and fibrous tissue responses.
The primary effects of mineralocorticoids (MC's) involve the
retention of certain minerals, particularly sodium, and the
elimination of potassium.
The most important and potent MC is aldosterone (ALDO); another
naturally occurring MC which is less potent is deoxycorticosterone
(DOC). If ALDO is present at abnormally high quantities (such as
following hemorrhage, bodily injury, or sodium deprivation), the
body will retain sodium and water, and will secrete potassium. This
can be a beneficial short-term response to stress. However, chronic
elevations of ALDO can be detrimental, such as in a patient with
heart failure who is suffering from edema (fluid accumulation), or
a patient with hypertension (high blood pressure). In patients with
edema or hypertension, an excess of ALDO promotes salt and water
retention and potassium loss, which are detrimental. Certain drugs,
most notably spironolactone and epoxymexrenone (discussed below),
can be used to suppress activity of elevated circulating ALDO, or
to suppress the synthesis of ALDO.
ALDO secretion is influenced by various signals involving
adrenocorticotropin hormone (ACTH), melanocyte stimulating hormone,
atrial natriuretic peptide, and plasma concentrations of sodium and
potassium, and by a multi-step pathway called the
renin-angiotensin-aldosterone (RAA) system. In response to certain
signals which indicate a low blood pressure, the kidneys secrete
renin, which cleaves a precursor peptide called angiotensinogen to
release a peptide having ten amino acid residues, called
angiotensin I. This peptide is cleaved by another enzyme called
angiotensin converting enzyme (ACE) to generate angiotensin II,
which has eight amino acid residues. In addition to being a potent
vasoconstrictor (which increases blood pressure), angiotensin II
functions as a hormone to stimulate the release of ALDO by zona
glomerulose cells in the adrenal cortex. The RAA system is
described in more detail in articles such as Weber and Brilla 1991
(full citations are provided below).
ALDO receptors (also called mineralocorticoid receptors (MCR or
MinR, or mineralosteroid receptors) are proteins that initially
reside in the cytoplasm of certain types of cells, such as smooth
muscle cells and fibroblasts in the aorta (see, e.g., Meyer and
Nichols 1981). When an ALDO receptor is activated by ALDO, the
receptor/ALDO complex (or lease some portion thereof) is
transported into the cell nucleus, where it binds to nuclear
chromatin and presumably causes an alteration in the transcription
of genes that encode proteins which are involved in the retention
of sodium and water by the body (see, e.g., Kornel et al. 1983).
For more information on ALDO receptors, see Agarwal and Lazar 1991
and additional references cited therein. Chemical methods of
synthesizing aldosterone are described in articles such as Barton
et al. 1975 and Miyano 1981.
FIBROSIS
Fibrosis (the generation of fibrotic tissue) is important in a
number of processes in adult mammals. In some processes, such as
wound healing, fibrosis is highly beneficial and essential to
survival. When one or more blood vessels, which function as
barriers to separate the intravascular and extracellular spaces,
are cut or otherwise broken or disrupted, an orderly wound healing
process is initiated. Certain types of blood cells such as
platelets release fibrinogen, plasminogen, and fibronectin into the
cellular interstitium; these molecules react with other molecules
to generate an extravascular coagulation and the formation of a
hydrophilic fibrin-fibronectin gel. Various growth factors are
believed to orchestrate the subsequent entry of immune and
inflammatory cells and fibroblasts into the gel and the formation
of new blood vessels within its interstices.
During the early stages, the gel is considered granulomatous
tissue. It is gradually absorbed and replaced by fibrous tissue.
One of the important components of such tissue is collagen, a
fibrous protein secreted by fibroblasts; it provides an
intercellular lattice or matrix which anchors cells in position in
cohesive tissue (such as muscle or blood vessels).
In addition to synthesizing and secreting collagen, fibroblasts
also synthesize and secrete collagenase, an enzyme which digests
collagen. In healthy tissue, the gradual cycle of collagen
secretion and degradation helps ensure that the protein fibers and
connective tissue remain flexible, elastic, and in a steady-state
concentration.
If fibrosis occurs as a wound-healing process in response to
injury, it is classified as "reparative" fibrosis. Similarly, if
fibrosis is initiated in an internal organ in response to the
necrosis of parenchymal cells (i.e., cells which are characteristic
of that particular organ, as distinct from non-specific cells), the
generation of fibrotic tissue constitutes reparative fibrosis. In
either case, collagen accumulation and connective tissue formation
usually resemble scar tissue.
In some situations which can generally be regarded as disease
conditions, blood vessels can lose their integrity and become
permeable to macromolecules, even in the absence of a cut or other
injury. In such situations, fibrosis can arise which is unwanted
and unnecessary; it resembles a wound healing response that has
gone awry. In the absence of parenchymal cell loss, this type of
fibrosis can be classified as a "reactive" fibrosis. If a
sufficient quantity of unwanted fibrotic tissue is generated in an
internal organ such as the heart, the fibrotic tissue can
compromise or seriously damage the functioning of the organ.
Humans or animals that suffer from chronic hypertension or edema
often are found, upon autopsy, to have heart muscle that suffers
from a characteristic often referred to by pathologists as "tough
beef." Instead of appearing pliable, elastic, and free of stranded
material, like a fresh high-quality filet mignon, the heart muscle
is riddled and interspersed with fibrotic strands which render the
heart muscle stiff and unable to flex, move, and function with full
efficiency. This condition is referred to as myocardial fibrosis
when it involves heart muscle (in medicine, the prefix "myo" refers
to muscle), or as cardiac fibrosis (which is somewhat broader,
since it can also include fibrosis in coronary arteries).
Myocardial fibrosis can be generated in lab animals in any of
several ways. Animals models which have been reported in the
literature (e.g., Doering et al. 1988 and Brilla et al. 1990)
involve: (1) renovascular hypertension (RHT), which can be induced
by surgically placing a constricting band around a renal artery for
a prolonged period (such as several weeks, in rats); and, (2)
ALDO-induced hypertension, in which animals are administered ALDO
(usually be means of a small osmotic pump implanted beneath the
skin, which slowly releases ALDO over a period of weeks) while
being fed a high-salt diet. Either of these interventions,
discussed in more detail below, will provoke an unwanted myocardial
fibrosis in a previously normal heart.
One of the main forms of myocardial fibrosis involves a condition
known as "left ventricular hypertrophy," which is discussed at some
length in Weber and Brilla 1991. The left ventricle is the largest
pumping chamber of the heart; it pumps blood through the entire
body and head, excluding the lungs (which receive blood from the
right ventricle). "Hypertrophy" refers to a non-tumorous increase
in the size of an organ or muscle, and in left ventricular
hypertrophy (LVH), the muscular wall of the left ventricle becomes
excessively large. LVH is the single most important risk factor
associated with adverse myocardial events, including myocardial
failure and sudden death.
In animal models, LVH can be induced (for the purpose of studying
it) by various means that generate hypertension over a prolonged
period of time. Such interventions include (1) surgically
constricting an artery that supplies the kidneys; (2) feeding
animals a high-sodium diet coupled with ALDO injections; and, (3)
infrarenal aortic banding, which involves surgically clamping the
abdominal aorta below the junction where the renal arteries branch
off; this reduces blood flow to the legs and elevates blood
pressure in the thoracic region. In many but not all cases, LVH is
accompanies by fibrosis (see, e.g., Jalil et al. 1988 and
1989).
Fibrosis has also been observed as a result of steroid use or abuse
by humans, and steroid administration to lab animals; see, e.g.,
Skelton 1954, Hassager et al. 1990, and Luke et al.
Most types of reactive fibrosis are initially characterized by
"perivascular" fibrosis (i.e., fibrosis which is localized around
blood vessels. As the process of fibrosis continues and fills the
spaces between various types of cells, it can be characterized as
an "interstitial" fibrosis.
Fibrosis is intimately related to collagen metabolism by fibroblast
cells. In the repair of wounds or cell necrosis, the activity of
fibroblasts in synthesizing fibrillar collagen and in secreting
collagenase will have a major impact on whether the fibrous tissue
component of the wound healing or tissue replacement response is
appropriate and proceeds to a successful conclusion. In reactive
fibrosis, the amount of undesired collagen deposition will also
depend on the level of activity of the fibroblasts involved.
Various factors are known to regulate collagen metabolism and
fibroblast growth, or both, and thereby govern collagen
accumulation (see, e.g., Rothe and Falanga 1989). These include
cytokines such as fibroblast growth factor, platelet derived growth
factor and transforming growth factor-beta.sub.1. Hormones are also
involved, such as the above-mentioned glucocorticoids, which oppose
various aspects of wound healing including inflammatory cell and
fibrous tissue responses and which have antifibrotic properties
relative to wound healing.
The processes of myocardial fibrosis, and the structure and
arrangement of collagen fibers and cells in heart muscle, are
described and illustrated in articles such as Weber and Brilla
1991. Articles which focus more specifically on the role of
cytokines and other growth factors on wound healing include
Blitstein-Willinger 1991 and Rothe and Falanga 1989.
ANTI-ALDOSTERONE DRUGS
A number of drugs have been identified which can inhibit the
activity of ALDO in the body, including spirolactones. The term
"spirolactone" indicates that a lactone ring (i.e., a cyclic ester)
is attached to another ring structure in a spiro configuration
(i.e., the lactone ring shares a single carbon atom with the other
ring). Spirolactones which are coupled to steroids are the most
important class of spirolactones from a pharmaceutical perspective,
so they are widely referred to in the pharmaceutical arts simply as
spirolactones. As used herein, "spirolactone" refers to a molecule
comprising a lactone structure coupled via a spiro configuration to
a steroid structure or steroid derivative.
One particular spirolactone which functions as an effective ALDO
antagonist is called spironolactone, which is marketed as an
anti-hypertensive and diuretic drug by G. D. Searle (Skokie, Ill.)
under the trademarks "Aldoctone" and "Aldactazide." Spironolactone
is the name commonly used by chemists; the full chemical name is
17-hydroxy-7-alpha-mercapto-3-oxo-17-alpha-pregn-4-ene-21-carboxylic
acid gamma-lactone acetate. This compound, its activities, and
modes of synthesis and purification are described in a number of
U.S. patents, including U.S. Pat. No. 3,013,012 (Cella and Tweit
1961) and 4,529,811 (Hill and Erickson 1985). Another
spironolactone effective as an aldosterone antagonist is
epoxymexrenone, which has the structural formula set forth below:
##STR1## Epoxymexrenone possesses high mineralocorticoid receptor
affinity (comparable to spironolactone) but with reduced binding
affinity for androgen and progesterone receptors. Initial studies
of this compound have demonstrated a Na.sup.+ /K.sup.+ effect
equipotent with spironolactone at a 50 mg dose.
Spironolactone and epoxymexrenone function as antagonists of ALDO;
they occupy ALDO receptors without triggering the normal receptor
activity. This competitive binding reaction reduces the ability of
ALDO molecules to bind to and trigger activity at such receptors.
As used herein, "aldosterone antagonist" refers to any compound
that suppresses the receptor-mediated activity of aldosterone; it
does not include compounds which reduce the amount of aldosterone
synthesized or secreted by the adrenal cortex, such as mespirenone
(discussed below).
When spironolactone and epoxymexrenone are used to suppress ALDO
activity, they promote the elimination of fluid and sodium by the
body, primarily via the kidneys and its formation of urine. Both of
these effects help control hypertension in people suffering from
high blood pressure. Spironolactone is therefore used to treat
hypertension, and epoxymexrenone has been demonstrated as effective
for this same purpose. The minimum effective anti-hypertensive
dosage of these spirolactone compounds in adults is about 50
milligrams (mg) per day; dosages often exceed this, and dosages of
200 to 400 mg/day are common for chronic treatment. Since
spironolactone and epoxymexrenone are metabolized and secreted
fairly rapidly, typical administration involves pills containing 25
to 100 mg, taken four times daily.
The anti-hypertensive dosage of aldosterone antagonists is
important to the subject invention, because it has been discovered
that spirolactones such as spironolactone and epoxymexrenone can be
used for an entirely different purpose (to inhibit fibrosis, as
described herein) at dosages that are below the dosages which have
anti-hypertensive effects. This is a useful finding, since it
indicates that spirolactones and other aldosterone antagonists can
be used to prevent unwanted fibrosis at dosages which have minimal
side effects and do not substantially alter the body fluids or
mineral concentrations of a patient.
As mentioned above, ALDO is secreted as one step in a multi-step
pathway involving renin and angiotensin, in the RAA system. Various
drugs have been identified which can inhibit one or more of the
steps in this pathway. For example, if a drug classified as an "ACE
inhibitor" (i.e., it inhibits angiotensin converting enzyme) is
used to suppress the formation of angiotensin II, secretion of ALDO
by the adrenal cortex will be suppressed. The most widely used ACE
inhibitor is captopril.
In addition, certain drugs have been identified which appear to
block ALDO synthesis and/or secretion by a more direct mechanism.
These drugs include 15,16-methylene spirolactone compounds called
Mespirenone (also called ZK 94679) and dethiolated Mespirenone
(also called ZK 91587); see Losert et al. 1986, Nickisch et al.
1991, and Agarwal and Lazar 1991).
One object of this invention is to disclose a method of using an
aldosterone antagonist, at a dosage which does not disrupt a
patient's normal electrolyte and water-retention balance, to treat
or prevent myocardial fibrosis. This and other objects will become
more apparent in the following summary and description.
SUMMARY OF THE INVENTION
This invention discloses a method of using an aldosterone
antagonist such as spironolactone, epoxymexrenone, or other
spirolactone, at a dosage which does not disrupt a patient's normal
electrolyte and water-retention balance, to inhibit myocardial
fibrosis, including left ventricular hypertrophy (LVH).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
This invention discloses a method of using an aldosterone
antagonist such as, for example, spironolactone, epoxymexrenone, or
other spirolactone, at a dosage which does not disrupt a patient's
normal electrolyte and water-retention balance, to prevent or
otherwise inhibit myocardial fibrosis. As discussed herein,
disruption of a patient's normal electrolyte balance refers to a
substantial alteration of sodium or potassium concentrations in the
patient's blood. At the dosages used to treat hypertension (high
blood pressure) or edema (excessive fluid accumulation in the
body), spironolactone and epoxymexrenone substantially reduce
sodium levels and increases potassium levels in the body. These
alternations in the homeostatic mineral balances of the body can
provoke a number of unpleasant side effects; accordingly, use of
lower dosages to inhibit myocardial fibrosis can avoid or minimize
such side effects.
As used herein, "treatment" or "inhibition" of myocardial fibrosis
are used interchangeably to include (1) treatment of patients in
which myocardial fibrosis has already reached dangerous or damaging
levels, and (2) preventive or prophylactic treatment of patients
who are at high risk of myocardial fibrosis, or who are displaying
symptoms that suggest the possible approach or onset of myocardial
fibrosis.
Patients can exhibit indications of myocardial fibrosis in any of
several ways, including symptoms reported by the patients (such as
shortness of breath), signs which are observed by a physician (such
as pleural effusion, which involves fluid in the chest), and
through the results of laboratory analyses (such as blood gas
abnormalities, or abnormal appearances on an echocardiogram). If a
physician or pathologist determines that myocardial fibrosis is
occurring or poses a serious threat in a specific patient, the
physician can prescribe an aldosterone antagonist such as a
spirolactone to inhibit the fibrosis.
An important aspect of this invention is that the treatment can
utilize a low dosage of the aldosterone antagonist. As described in
Example 2, tests have indicated that spironolactone is effective in
preventing myocardial fibrosis at dosages which are below
anti-hypertensive dosages. This allows anti-fibrotic administration
of an aldosterone antagonist at dosages which have minimal side
effects and do not substantially disrupt electrolyte balances or
water retention in the patient.
The evidence which shows that ALDO is a primary and direct
causative agent which induces myocardial fibrosis is contained in
Examples 1 through 3. Example 1 describes three different
techniques that were used to create hypertension in rats:
(1) in some rats, renovascular hypertension (RHT) was induced by
surgically placing a constricting band around the right renal
artery, to induce unilateral renal ischemia. This method is
described in Doering et al. 1988. The right kidney, in response to
the apparent low blood pressure, releases renin, which activates
the renin-angiotensin-aldosterone (RAA) system. This hormonal
complex results in increased blood pressure, increased angiotensin
II concentrations, and increased ALDO concentrations.
(2) infrarenal banding (IRB) was used in other rats. This involves
constricting flow through the aorta, below the junction where the
renal arteries (which supply the kidneys) branch off from the
abdominal aorta. This reduces blood flow to the legs, and it
elevates blood pressures in the kidneys and other internal organs,
excluding the lungs (which are served by the right ventricle).
Despite the rise in blood pressure, concentrations of circulating
angiotensin II and ALDO remain relatively normal in this
treatment.
(3) in other rats, direct infusion of ALDO was used, via osmotic
minipumps. These pumps do not use moving parts; instead, they act
by causing a solution to diffuse out of the pump through a
membrane, driven by osmotic pressure. These pumps, loaded with
ALDO, were implanted subcutaneously in uninephrectomized rats
(i.e., rats from which one kidney had been removed), and they
released ALDO directly into the circulating blood. After ALDO
levels increased, due to this direct ("primary") infusion, the
levels of angiotensin II typically decreased during the first four
weeks, then gradually returned to normal levels.
These treatments lasted 8 weeks; during this time, all three
treatment groups showed comparable levels of elevated blood
pressure. The animals were then sacrificed and the hearts were
dissected and analyzed. All three groups showed comparable levels
of left ventricular hypertrophy. However, increased collagen
formation (indicating unwanted fibrosis) was found only in rats
with renovascular hypertension (which causes elevated
concentrations of both angiotensin II and ALDO) and in rats
receiving ALDO by minipump. The rats that were treated with
infrarenal banding (with normal levels of angiotensin II and ALDO)
developed left ventricular hypertrophy, but they did not suffer a
fibrotic response.
These findings indicated that myocardial fibrosis involved a
cellular mechanism that did not depend solely on the presence of
hypertension, which apparently was mediated by some combination of
angiotensin II and/or aldosterone. These results, which were
reported in Doering et al. 1988 and Brills et al. 1990, began the
process of identifying the role of mineralocorticoids in promoting
fibrosis, but they did not adequately resolve a number of
questions, such as questions relating to the respective
contributions of those two hormones. To further resolve those and
other issues, additional tests were done involving (1) the in vivo
use of captopril, an ACE inhibitor which suppresses the synthesis
of angiotensin II, and (2) in vitro tests involving cultured
fibroblast cells.
The tests involving captopril are described in Example 2. Briefly,
rats were subjects to either of two treatments: (1) direct
injection of ALDO coupled with a high salt diet, in
uni-nephrectomized rats, or (2) unilateral renal ischemia, to
induce renovascular hypertension (RHT). These two treatments can be
regarded as primary ALDO elevation (i.e., direct injection of
ALDO), or secondary ALDO elevation (due to RHT treatment, which
increases ALDO levels as one step in the RAA hormonal cascade).
Each treatment group (primary or secondary) was divided into four
subgroups which received different treatments over an eight week
period, as follows:
(1) One pair of subgroups received an ACE inhibitor, captopril, to
suppress the synthesis of angiotensin-II in those with secondary
ALDO elevation;
(2) One pair of subgroups received a low does of spironolactone, an
ALDO antagonist. Under the conditions used, this dosage was not
high enough to suppress hypertension in animals with primary ALDO
elevation.
(3) One pair of subgroups received a high dose of spironolactone,
sufficient to suppress hypertension in animals with primary ALDO
elevation.
(4) The control subgroups did not receive any treatment, other than
the treatments which induced primary or secondary ALDO
elevation.
The results were as follows:
(1) Captopril treatment, which inhibited the formation of
angiotensin-II, prevented hypertension in both subgroups. It also
prevented secondary hyperaldosteronism and fibrosis in rats with
induced RHT. However, it did not prevent fibrosis in rats treated
directly with ALDO infusion.
(2) The high dose of spironolactone prevented hypertension, LVH,
and fibrosis. The small does of spironolactone was not able to
prevent either hypertension or LVH, but it was able to prevent
fibrosis.
These findings indicated that ALDO, rather than angiotensin or
arterial hypertension, is a primary and direct causative agent of
fibrosis. They also indicated that fibrosis can be blocked by
suppressing activity at ALDO receptors, using an ALDO antagonist
such as spironolactone or epoxymexrenone, at a dosage below the
dosage required to suppress hypertension.
In the in vitro tests described in Example 3, cardiac fibroblast
cells were harvested from rats, separated from each other using
collagenase, and divided into different treatment groups. One group
of cells was incubated with aldosterone. A second group of cells
was incubated with dexamethasone, a glucocorticoid that inhibits
collagen synthesis. A third group was incubated with a mixture of
aldosterone and spironolactone, and a fourth (control) group was
not treated with any exogenous gluco- or mineralocorticoids. During
a 24-hour incubation period, the nutrient medium contained proline
(an amino acid) which was radiolabelled with tritium [.sup.3 H].
Since proline is present at high concentrations in collagen, .sup.3
H-proline incorporation into insoluble protein during the
incubation period provided an indicator of collagen synthesis.
At the end of the incubation period, cells were lysed and insoluble
proteins were purified using chemical processing followed by
centrifugation. The pelleted proteins were resuspended and then
digested with collagenase, to solubilize collagen and its amino
acids. The mixture was pelleted again and then analyzed to
determine the concentration of solubilized labelled protein in the
supernatant. This quantity was divided by the total labelled
proline content in both the pellet and the supernatant, to provide
a numerical index of collagen formation.
The results, shown in Table 1 (in Example 3), indicate that ALDO
caused a marked increase in collagen synthesis in cardiac
tissue.
ORAL DOSAGES AND REDUCTION OF SIDE EFFECTS
Spironolactone is sold in 25, 50 or 100 mg tablets, which are taken
orally for edema or hypertension. It is sold by G. D. Searle and
Company (Skokie, Ill.) under the trademarks Aldoctone (containing
spironolactone as the sole active ingredient) and Aldactazide
(which contains spironolactone combined with hydrochlorothiazide, a
diuretic).
The Physician's Desk Reference (PDR) makes a number of pertinent
comments about dosages for spironolactone when treating edema,
hypertension. For "essential hypertension" in adults, initial
dosages of 50 to 100 mg are recommended; for edema (including
congestive heart failure, hepatic cirrhosis, and hephrotic
syndrome) initial dosages of 100 are recommended. In either
situation the patient should be monitored and the dosages should be
adjusted, depending on how the patient responds, to a range of 25
to 100 mg (or more). For a somewhat different condition called
hypokalemia (potassium deficiency, usually induced by treatment
with a diuretic), dosages of 25 to 100 mg are also recommended.
These recommended dosages must be viewed in light of warnings in
the PDR about the risks and unwanted side effects of
spironolactone. It has been shown to be a tumorigen (cancer-causing
agent) in chronic toxicity studies; in addition, "Carcinoma of the
breast has been reported in patients taking spironolactone, but a
cause and effect relationship has not been established." Comparable
risks and side effects exist for other aldosterone antagonists such
as epoxymexrenone.
In addition to possibly increasing the risk of cancer,
spirolactones such as spironolactone and epoxymexrenone cause major
disruptions in the balance of minerals within the body. These
disruptions can create or aggravate any number of unpleasant
symptoms that are given labels such as hyperkalemia, hyperchloremic
metabolic acidosis, hyponatremia, gynecomastia, and
agranulocytosis. In addition, as stated in the PDR, "Other adverse
reactions that have been reported in association with
[spironolactone] are: gastrointestinal symptoms including cramping
and diarrhea, drowsiness, lethargy, headache, maculopapular or
erythematous cutaneous eruptions [i.e., spontaneous or excessive
bleeding], urticaria [i.e., intense itching], mental confusion,
drug fever, ataxia [i.e., loss of muscle coordination], inability
to achieve or maintain erection, irregular menses [menstruation] or
amenorrhea, postmenopausal bleeding, hirsutism [i.e., abnormal hair
growth], depending of the voice, gastric bleeding, ulceration,
gastritis, and vomiting."
Any one of the above-listed side effects can make life miserable
for a patient who must endure it. The fact that all of these side
effects have been associated with spironolactone and epoxymexrenone
indicates that such spirolactones and other aldosterone antagonists
can have unpleasant side effects, and should be taken only when
necessary and at the lowest dosage that can achieve the necessary
result.
The risks, rates of occurrence, and severity of these noxious side
effects will be substantially reduced if the dosages of aldosterone
antagonist can be reduced. Due to a number of factors, even a
modest reduction in dosage can completely eliminate or greatly
reduce such side effects in many people. Therefore, the discovery
that aldosterone antagonists such as spironolactone and
epoxymexrenone are effective against myocardial fibrosis, at
dosages lower than necessary for controlling hypertension, is an
important discovery and an important feature of this invention. In
particular, it has been found that aldosterone antagonists, and
especially spirolactones such as spironolactone and epoxymexrenone
can be administered in dosages which are effective for preventing
myocardial fibrosis but insufficient to substantially increase
sodium excretion or substantially reduce potassium retention. This
is demonstrated by the evidence that myocardial fibrosis is
inhibited at dosages which are insufficient to affect blood
pressure, since sodium excretion is not substantially increased or
potassium retention substantially reduced by administration of an
aldosterone antagonist unless the dosage is sufficient to lower
arterial blood pressure.
In accord with this teaching, this invention discloses oral tablets
or capsules containing from 10 to 20 mg of spironolactone or
epoxymexrenone. In view of the noxious side effects of these
spirolactones, the difference between this dosage range and the
smallest dosages currently available (25 mg) is substantial, and
will provide a major benefit to many users.
Spirolactones such as Spironolactone and epoxymexrenone are
discussed herein because they are archetypical ALDO antagonists,
which selectively and specifically block ALDO receptors. If
desired, other ALDO antagonists could be used instead of
spironolactone, either to block the activity of ALDO molecules at
ALDO receptors, or to suppress the biosynthesis of ALDO at the
adrenal cortex. Since the activation of ALDO receptors by ALDO
molecules has been shown to be a key step in the chain of events
leading to myocardial fibrosis, any ALDO antagonist which can block
or inhibit that ALDO/receptor interaction will effectively break
that chain and stop the cascade of events leading to myocardial
fibrosis.
EXAMPLES
Example 1
In Vivo Studies Involving Chronic Hypertension
Arterial hypertension was induced in eight week old male Sprague
Dawley rats (180-200 g), using three different techniques:
(1) in some animals, renovascular hypertension (RHT) was induced by
surgically placing a constricting band around the right renal
artery, to induce unilateral renal ischemia.
(2) in other animals, infrarenal banding (IRB) was used to
mechanically constrict blood flow through the aorta below the
junction where the renal arteries branch off. This elevates blood
pressures in the kidneys, but angiotensin II and ALDO remain
relatively normal.
(3) ALDO (d-aldosterone, purchased from Sigma Chemical, St. Louis,
Mo.) was directly infused into the animals at the rate of 0.75
micrograms (ug) per hour, via osmotic minipumps (Alzet Model 2002),
Alza Corp., Palo Alto, Calif.) which were implanted subcutaneously
in uni-nephrectomized rats. These rats were fed standard rat chow
with a sodium concentration of 0.4% and additional sodium in the
drinking water (10 g/L) resulting in a high sodium diet. ALDO
levels were elevated due to the direct ("primary") infusion.
Angiotensin II levels typically decreased (to about 10-015
micrograms per ml of blood) during the first four weeks, then
gradually rose to normal levels (about 30 pg/ml).
Control animals were (1) uninephrectomized rats on a high sodium
diet with implanted minipumps, but where aldosterone administration
was withheld, and (2) unoperated, untreated, age and sex matched
controls.
These treatments lasted for 8 weeks. During this period, the rats
were monitored for hypertension, using a tail cuff. All three
treatment groups suffered comparable levels of hypertension (in the
range of about 190-200 mm Hg) compared to blood pressures of about
130-140 in control animals. Plasma aldosterone was measured using a
radioimmunoassay (Diagnostic Products Corp., Los Angeles, Calif.)
with sample aldosterone competing with .sup.125 I-radiolabelled
aldosterone for antibody sites. Urine sodium and potassium
concentrations were measured by flame photometry.
At the end of the 8 week treatment, the rats were sacrificed, and
the hearts were dissected and analyzed. Coronal sections were
dehydrated and embedded in paraffin. The weights of the left
ventricles were determined and compared to right ventricle weight
and total body weight. Thereafter, sections which were 5 microns
thick, containing a complete cross sectional cut of the left
ventricle, were stained with the collagen specific dye Sirius Red
F3BA (Pfaltz & Bauer, Stamford Conn.). Connective tissue and
muscle areas were identified according to their gray level, where
collagen fibers appear black, myocytes are gray, and interstitial
space is white. Digitized profiles were created using an automated
image analyzer (Quantimet 520, Cambridge Instruments, Inc.,
Deerfield, Ill.) and transferred to a computer that calculated
collagen volume fraction as the sum of all connective tissue areas
in the entire section, divided by the sum of all connective tissue
and muscle areas. Perivascular collagen was excluded from this
analysis and was measured separately. Total collagen volume
fraction (including all perivascular collagen), as determined by
this morphometric approach, is closely related to hydroxyproline
concentration of the left ventricle.
Similarly, perivascular collagen area (PVCA) normalized to vessel
luminal area of intramural coronary arteries was determined in
Sirius Red stained tissue using the automated image analyzer. Only
those intramyocardial vessels which appeared circular on cross
section were analyzed to ensure correct normalization for vessel
luminal area; on the average there was 15 such vessels found in the
left ventricle. The investigator responsible for the morphometric
analysis was blinded as to each experimental group.
Interstitial collagen volume fraction (CVF) was determined using
picrosirius stained tissue.
All three treatment groups showed comparable levels of arterial
hypertension and left ventricular hypertrophy. However, increased
CVF indicating fibrosis was found only in rats with renovascular
hypertension (RHT), which causes elevated concentrations of both
angiotensin II and ALDO, and rats receiving ALDO by minipump. The
rats that were treated with infrarenal banding (with normal levels
of angiotensin II and ALDO) suffered left ventricular hypertrophy,
but they did not suffer a fibrotic response. This finding indicated
that cardiac fibrosis apparently involved a cellular mechanism that
did not depend solely on the presence of hypertension, which
apparently was mediated by some combination of angiotensin II
and/or aldosterone.
Example 2
Studies Involving Inhibition of Angiotensin II
Rats were treated to generate primary hyperaldosteronism (by
directly injecting ALDO in uninephrectomized rats) or secondary
hyperaldosteronism (by creating unilateral renal ischemia, which
induced RHT as above) using the same procedures described in
Example 2. Each treated group was divided into four subgroups which
received different treatment over an eight week period, as
follows:
(1) One pair of subgroups received an ACE inhibitor, captopril, to
suppress the synthesis of angiotensin-II.
(2) One pair of subgroups received a relatively low dose (20
mg/kg/day) of the ALDO antagonist, spironolactone. This dosage was
not sufficient to suppress hypertension.
(3) One pair of subgroups received a higher quantity (200
mg/kg/day) of spironolactone, which is sufficient to suppress
hypertension.
(4) The control subgroups did not receive any treatment, other than
the treatments which induced primary or secondary ALDO
elevation.
The results were as follows:
(1) Captopril treatment, which inhibited the formation of
angiotensin-II, prevented hypertension in both subgroups. It also
prevented the induction of secondary hyperaldosteronism, and the
induction of fibrosis, in rats with induced RHT. However, it did
not prevent fibrosis in rats that were treated directly with ALDO
injections.
(2) The large does of spironolactone prevented hypertension, LVH,
and fibrosis. The small does of spironolactone was not able to
prevent either hypertension or LVH, but it was able to prevent
fibrosis.
These findings indicated that ALDO, rather than angiotensin or
arterial hypertension, is the primary and direct causative agent of
fibrosis. They also indicate that fibrosis can be blocked by
suppressing activity at ALDO receptors, using spironolactone.
Example 3
In Vitro Studies of Fibroblasts
Cardiac fibroblast cells were harvested from rats, separated by
digestion with collagenase, and divided into different treatment
groups which were cultured in nutrient medium supplemented with
fetal calf serum. One group was incubated with 10.sup.-9 M
aldosterone; another group was incubated with 10.sup.-9 M
dexamethasone, a glucocorticoid which inhibits collagen synthesis.
A third group was incubated with a mixture of 10.sup.-9 M
aldosterone and 10.sup.-6 M spironolactone. A fourth (control)
group was not treated with any exogenous gluco- or
mineralocorticoids.
During the 24-hour incubation period, the nutrient medium contained
proline which was radiolabelled with tritium [.sup.3 H]. Since
proline is present at high concentrations in collagen, .sup.3
H-proline incorporation into soluble protein during the incubation
period provided an indicator of collagen synthesis (CS). At the end
of the incubation period, the cells were lysed and insoluble
proteins were purified using chemical processing followed by
centrifugation. The pelleted proteins were resuspended and then
digested with collagenase, to solubilize the collagen. The mixture
was pelleted again and then analyzed to determine the concentration
of solubilized labelled protein in the supernatant. This quantity
was divided by the total labelled proline content in both the
pellet and the supernatant, to provide a numerical index of
collagen formation. This number has been shown to correlate well
with the digital optical analysis of stained tissue described in
Example 1.
Cell counts were estimated visually on a number per square
centimeter basis; since they are anchorage dependent, they cannot
be analyzed easily using flow cytometry. DNA synthesis was analyzed
using radiolabelled thymidine and an intercalating dye,
Fluorochrome H33258 (Calbiochem, La Jolla, Calif.).
The results, shown in Table 1, indicate that ALDO caused a marked
increase in collagen synthesis.
TABLE 1 ______________________________________ Treatment
C#/10.sup.5 CS/cell CS/DNA CS/protein
______________________________________ ALDO 2.3 .+-. 0.4 16.1 .+-.
3.9 3.3 .+-. 0.7 5.9 .+-. 1.0 DEXA 2.4 .+-. 0.5 1.6 .+-. 0.7 0.4
.+-. 0.2 1.5 .+-. 0.8 ALDO + SL 1.9 .+-. 0.5 18.2 .+-. 4.2 2.9 .+-.
0.2 6.1 .+-. 0.8 Control 2.1 .+-. 0.3 5.6 .+-. 2.2 0.8 .+-. 0.2 2.9
.+-. 0.5 ______________________________________
Thus there has been shown and described a method and a composition
for using aldosterone antagonists to suppress undesired myocardial
fibrosis, at a dosage below the level required to suppress
hypertension. This disclosure is set forth in certain specific
embodiments, but it will be apparent to those skilled in the art
that various changes and modifications to the specific embodiments
described herein are possible. Any such changes that do not depart
from the spirit and scope of the invention are deemed to be covered
by the invention, which is limited only by the claims which
follow.
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